The Type III Effector XopC from //Xanthomonas//

Author: Alice Boulanger
Internal reviewer: Ralf Koebnik

Class: XopC
Families: XopC1 and XopC2
Prototype (XopC1): XCV2435 (Xanthomonas euvesicatoria pv. euvesicatoria, ex Xanthomonas campestris pv. vesicatoria; strain 85-10)
GenBank ID (XopC1): CAJ24112.1 (834 aa)
Prototype (XopC2): XOC_1264 (Xanthomonas oryzae pv. oryzicola ; strain BLS256)
GenBank ID (XopC2): AEQ95452.1 (549 aa - likely too short)
GenBank ID (XopC2; strain GX01): QEO98660.1 (596 aa)
RefSeq ID (XopC1): WP_011347616.1 (834 aa)
RefSeq ID (XopC2): WP_041183113.1 (412 aa - likely too short)
3D structure: Unknown. XopC2 from Xanthomonas axonopodis pv. punicae is predicted to be a 661 amino-acids protein with 5 alpha helices and 17 beta strands. It has 21 protein binding and one helical transmembrane region of 18 amino acids (Mondal et al., 2020).

XopC was discovered in X. campestris pv. vesicatoria (Xcv) in a cDNA-AFLP screen (Noël et al., 2001). XopC was also identified in a genetic screen, using a Tn5-based transposon construct harboring the coding sequence for the HR-inducing domain of AvrBs2, but devoid of the effectors' T3SS signal, that was randomly inserted into the genome of Xcv strain 85-10. The XopC::AvrBs2 fusion protein triggered a Bs2-dependent hypersensitive response (HR) in pepper leaves (Roden et al., 2004).

A chimeric protein consisting of XopC fused to a c-myc epitope (first 466 amino acids plus 5 kDa epitope) was secreted into culture supernatants of a strain with a constitutively active form of HrpG in a type III secretion-dependent manner (Noël et al., 2003). Another chimeric protein consisting of XopC fused to an N-terminally deleted derivative of the effector protein AvrBs3 (XopC₂₀₀-AvrBs3_∆2-153) was used to assay the translocation of XopC into plant cells (Noël et al., 2003). AvrBs3_∆2-153 was no longer delivered by the T3SS but was still able to induce the HR response in the pepper cultivar ECW-30R when artificially delivered by Agrobacterium (Szurek et al., 2002). XopC₂₀₀-AvrBs3_∆2-153 was detected in supernatant of a strain with a constitutively active form of hrpG in a type III secretion-dependent manner. Translocation of this chimeric protein into plant cells was confirmed by the observation of HR obtained on pepper cultivar ECW-30R.

Type III-dependent secretion was also confirmed using a calmodulin-dependent adenylate cyclase reporter assay, with a ΔhrpF mutant strain serving as negative control (Roden et al., 2004).

Translocation of the XopC::AvrBs3 chimeric protein was independent of the export control protein, HpaC (Büttner et al., 2006).

The xopC gene was shown to be expressed in a hrpG- and hrpX-dependent manner. No PIP box was identified in the promoter region (Noël et al., 2001; Noël et al., 2003).

qRT-PCR revealed that transcript levels of 15 out of 18 tested non-TAL effector genes (as well as the regulatory genes hrpG and hrpX), including xopC, were significantly reduced in the Xanthomonas oryzae pv. oryzae ΔxrvC mutant compared with those in the wild-type strain PXO99^A (Liu et al., 2016).

• A deletion of xopC did not affect pathogenicity or bacterial growth in plants (Noël et al., 2003).
• Roden et al. did not find significant growth defects of a Xcv ΔxopC mutant in susceptible pepper and tomato leaves (Roden et al., 2004)
• Later, 86 Solanaceae lines mainly of the genus Nicotiana were screened for phenotypical reactions after Agrobacterium tumefaciens-mediated transient expression of 21 different Xcv effectors. Transient expression of XopC exclusively induced plant reactions in lines of the genus Solanum (Adlung et al., 2006). Xcv 85-10 strain deleted for xopC induced weaker reactions than the wild type in S. americanum, which could be complemented by ectopic expression of xopC. Deletion of xopC did not affect visible reactions in N. benthamiana and N. tabacum to infection with Xcv. Thus, XopC contributes to Xcv-induced phenotypes in certain non-host plants (Adlung et al., 2006).
• The absence of xopC in the genome of Xcv led to an accelerated AvrBs1-induced HR in resistant pepper plants, if the plants were additionally stressed by exogenous application of salicylic acid (SA). This phenotype was complemented by xopC, but not by a xopC derivative carrying a mutation in the predicted HAD-like hydrolase sequence (Herzfeld, 2013).
• Virus-induced gene silencing (VIGS) of OAS-TL in planta abolished the acceleration of AvrBs1-mediated HR formation induced by the absence of xopC in Xcv in resistant pepper plants dependent on SA. These data suggest, that the induction of the AvrBs1-dependent HR in resistant pepper plants is SA-stress dependently delayed by XopC, which is reliant on a HAD-like hydrolase domain in XopC. This delay is mediated by the XopC plant interaction partner OAS-TL. Furthermore, expression analysis showed an increased accumulation of β-1,3-Glucanase transcript in Xcv-infected, resistant pepper plants by the presence of xopC. These findings indicated that XopC influences different mechnisms of the plant metabolism (Herzfeld, 2013).
• XopC2 of X. citri pv. punicae was found to contribute to the bacterial blight development on pomegranate fruit plants. Xap ΔxopC2 was demonstrated to cause reduced the blight lesions when inflitrated on pomegranate leaves, induce defense responses like callose deposition, ROS production and upregulate immune-responsive genes in its natural host plants (Mondal et al., 2020).
• Ectopic expression of XopC2 was found to promote jasmonate signaling and stomatal opening in transgenic rice plants, which were more susceptible to X. oryzae pv. oryzicola infection (Wang et al., 2021).

XopC localises to the plant cell cytoplasm (Mondal et al., 2020) and the nucleus (Herzfeld, 2013).

XopC contains a predicted phosphoribosyl transferase domain and a putative haloacid dehalogenase (HAD)-like hydrolase domain in its C-terminal end. Phenotype of point mutation in catalytic domain have shown that HAD-like hydrolase activity is required for the XopC deleterious effect in yeast (Salomon et al., 2011). XopC2 represents a family of atypical kinases that specifically phosphorylate OSK1, a universal adaptor protein of the Skp1-Cullin-F-box ubiquitin ligase complexes (Wang et al., 2021).

Yeast-2-hybrid studies revealed a XopC interactor, which also interacted with XopC in planta. The interactor localises to the plant cell cytoplasm and carries typical features of plant cytosolic O-acetylserine (thiol)lyases (OAS-TL). It shows OAS-TL activity in vivo and in vitro. The latter one is enhanced by adding XopC (Herzfeld, 2013).

Close, full-length homologs (>90% sequence identity) of XopC1 have only been found in several strains of clade-2 xanthomonads, such as X. citri, X. euvesicatoria, X. fragariae, X. gardneri, X. hortorum, and X. phaseoli (BLASTP and TBLASTN performed in June 2020).

The distantly related XopC2 has homologs in X. citri, X. axonopodis, X. euvesicatoria, X. oryzae, X. phaseoli, and X. translucens (BLASTP and TBLASTN performed in June 2020)

XopC1: Ralstonia solanacearum (RipC2), Trinickia caryophylli (Paraburkholderia caryophylli), Xylophilus ampelinus (BLASTP and TBLASTN performed in June 2020).

XopC2: Acidovorax ssp., Pseudomonas cissicola, Ralstonia solanacearum (RipC1) (BLASTP and TBLASTN performed in June 2020).

Close, full-length homologs (>90% sequence identity) of XopC1 have only been found in several strains of clade-2 xanthomonads, such as X. citri, X. euvesicatoria, X. fragariae, X. gardneri, X. hortorum, and X. phaseoli (BLASTP and TBLASTN performed in June 2020).

The distantly related XopC2 has homologs in X. citri, X. axonopodis, X. euvesicatoria, X. oryzae, X. phaseoli, and X. translucens (BLASTP and TBLASTN performed in June 2020)

XopC1: Ralstonia solanacearum (RipC2), Trinickia caryophylli (Paraburkholderia caryophylli), Xylophilus ampelinus (BLASTP and TBLASTN performed in June 2020).

XopC2: Acidovorax ssp., Pseudomonas cissicola [a pathovar of Xanthomonas citri], Ralstonia solanacearum (RipC1) (BLASTP and TBLASTN performed in June 2020).

Adlung N, Prochaska H, Thieme S, Banik A, Blüher D, John P, Nagel O, Schulze S, Gantner J, Delker C, Stuttmann J, Bonas U (2006). Non-host resistance induced by the Xanthomonas effector XopQ is widespread within the genus Nicotiana and functionally depends on EDS1. Front. Plant Sci. 30: 1796. DOI: 10.3389/fpls.2016.01796

Büttner D, Lorenz C, Weber E, Bonas U (2006). Targeting of two effector protein classes to the type III secretion system by a HpaC- and HpaB-dependent protein complex from Xanthomonas campestris pv. vesicatoria. Mol Microbiol. 59: 513-527. DOI: 10.1111/j.1365-2958.2005.04924.x

Herzfeld EM (2013). Identifizierung und Charakterisierung von dem pflanzlichen Interaktionspartner OAS-TL des Typ-III-Effektors XopC. Doctoral Thesis, Martin-Luther-Universität Halle-Wittenberg, Germany. PDF: opendata.uni-halle.de/handle/1981185920/7783

Liu Y, Long J, Shen D, Song C (2016). Xanthomonas oryzae pv. oryzae requires H-NS-family protein XrvC to regulate virulence during rice infection. FEMS Microbiol. Lett. 363: fnw067. DOI: 10.1093/femsle/fnw067

Mondal KK, Soni M, Verma G, Kulshreshtha A, Mrutyunjaya S, Kumar R ( 2020). Xanthomonas axonopodis pv. punicae depends on multiple non-TAL (Xop) T3SS effectors for its coveted growth inside the pomegranate plant through repressing the immune responses during bacterial blight development. Microbiol Res. 240: 126560 DOI: 10.1016/j.micres.2020.126560

Noël L, Thieme F, Gäbler J, Büttner D, Bonas U (2003). XopC and XopJ, two novel type III effector proteins from Xanthomonas campestris pv. vesicatoria. J. Bacteriol. 185: 7092-7102. DOI: 10.1128/jb.185.24.7092-7102.2003

Noël L, Thieme F, Nennstiel D, Bonas U (2001). cDNA-AFLP analysis unravels a genome-wide hrpG-regulon in the plant pathogen Xanthomonas campestris pv. vesicatoria. Mol. Microbiol. 41: 1271-1281. DOI: 10.1046/j.1365-2958.2001.02567.x

Roden JA, Belt B, Ross JB, Tachibana T, Vargas J, Mudgett MB (2004). A genetic screen to isolate type III effectors translocated into pepper cells during Xanthomonas infection. Proc. Natl. Acad. Sci. USA 101: 16624-16629. DOI: 10.1073/pnas.0407383101

Salomon D, Dar D, Sreeramulu S, Sessa G (2011). Expression of Xanthomonas campestris pv. vesicatoria type III effectors in yeast affects cell growth and viability. Mol. Plant-Microbe Interact. 24: 305-314. DOI: 10.1094/MPMI-09-10-0196

Szurek B, Rossier O, Hause G, Bonas U (2002). Type III-dependent translocation of the Xanthomonas AvrBs3 protein into the plant cell. Mol. Microbiol. 46: 13-23. DOI: 10.1046/j.1365-2958.2002.03139.x

Wang S, Li S, Wang J, Li Q, Xin XF, Zhou S, Wang Y, Li D, Xu J, Luo ZQ, He SY, Sun W (2021). A bacterial kinase phosphorylates OSK1 to suppress stomatal immunity in rice. Nat. Commun.12: 5479. doi: 10.1038/s41467-021-25748-4

This fact sheet is based upon work from COST Action CA16107 EuroXanth, supported by COST (European Cooperation in Science and Technology).